An Investigator-Initiated Open-Label Trial of Sonidegib in Advanced Basal Cell Carcinoma Patients Resistant to Vismodegib Christina Danial, Kavita Y

Published OnlineFirst November 6, 2015; DOI: 10.1158/1078-0432.CCR-15-1588

Clinical Trial Brief Report Clinical Cancer Research An Investigator-Initiated Open-Label Trial of Sonidegib in Advanced Basal Cell Carcinoma Patients Resistant to Vismodegib Christina Danial, Kavita Y. Sarin, Anthony E. Oro, and Anne Lynn S. Chang

Abstract

Purpose: To assess the tumor response to the smoothened sive disease with sonidegib. Three patients experienced stable (SMO) inhibitor, sonidegib (LDE225), in patients with an disease and discontinued sonidegib either due to adverse events advanced basal cell carcinoma (BCC) resistant to treatment with (n ¼ 1) or due to election for surgery (n ¼ 2). The response of one vismodegib (GDC0449). patient was not evaluable. SMO mutations with in vitro data Experimental Design: Nine patients with an advanced suggesting resistance to Hh pathway inhibition were identiﬁed BCC that was previously resistant to treatment with vismode- in 5 patients, and none of these patients experienced responses gib were given sonidegib in this investigational, open- while on sonidegib. label study. Tumor response was determined using the Conclusion: Patients with advanced BCCs that were response evaluation criteria in solid tumors. SMO mutations previously resistant to treatment with vismodegib similarly were identiﬁed using biopsy samples from the target BCC demonstrated treatment resistance with sonidegib. Patients location. who have developed treatment resistance to an SMO inhibitor Results: The median duration of treatment with sonidegib was may continue to experience tumor progression in response to 6 weeks (range, 3–58 weeks). Five patients experienced progres- other SMO inhibitors. Clin Cancer Res; 1–5. 2015 AACR.

Introduction Sonidegib (LDE225) is a new SMO inhibitor approved in 2015 by the FDA for locally advanced BCCs. Clinical trials have shown Smoothened (SMO) inhibitors are a new class of drugs for the its efficacy against locally advanced BCCs (6, 7). Sonidegib blocks treatment of advanced basal cell carcinomas (BCC) that include hedgehog signaling by selective inhibition of SMO, even though locally advanced and metastatic tumors (1). Vismodegib its chemical structure is different from vismodegib (8). Although (GDC0449) is a first-in-class SMO inhibitor approved by the antitumor activity has been reported in patients with advanced FDA for the treatment of both locally advanced and metastatic BCCs who were treated with sonidegib, an in vitro study has shown BCCs in 2012 (1, 2). Although patients treated with vismodegib that cells with SMO mutations display resistance to sonidegib can have partial and complete responses, more than 50% of (4, 6, 7). The purpose of this investigator initiated open-label patients are refractory to treatment, whereas over 20% of initial study was to assess the tumor response to sonidegib in patients responders develop resistance and experience disease progression with an advanced BCC that previously was resistant to treatment or recurrence (2, 3). Recently, a subset of mutations in the drug with vismodegib. target, SMO, has been identified in advanced BCCs resistant to vismodegib (4, 5). Acquired SMO mutations maintain hedgehog signaling by either impairing drug binding or inducing constitu- Materials and Methods tive activity of SMO, resulting in resistance to vismodegib (4, 5). Study patients These SMO mutations have also been shown to display functional Approval was obtained from the Institutional Review Board, resistance to vismodegib in vitro (4). The clinical outcome of a and written informed consent was obtained from all participants. chemically distinct SMO inhibitor on an advanced, vismodegib- An open-label study was conducted at a single academic institu- resistant BCC in vivo is unknown. tion from October 2011 to December 2013. This study was registered as NCT01529450 on clinicaltrials.gov.

Department of Dermatology, Stanford University School of Medicine, Inclusion criteria Stanford, California. Eligible patients were those with advanced (locally advanced or Note: Supplementary data for this article are available at Clinical Cancer metastatic) BCC that clearly demonstrated either primary or Research Online (http://clincancerres.aacrjournals.org/). secondary treatment resistance with vismodegib, as documented Corresponding Author: Anne Lynn S. Chang, Stanford University School of by their previous treating physician. Composite response end- Medicine, 450 Broadway Street Pavilion C, 2nd Floor MC 5334, Redwood City, point incorporating RECIST 1.0 (1) had been used to quantita- CA 94063. Phone: 650-721-7151; Fax: 650-721-3464; E-mail: tively define resistance to vismodegib (Supplementary Fig. S1). [email protected] Primary resistance was defined as stable or progressive disease; doi: 10.1158/1078-0432.CCR-15-1588 secondary resistance was defined as progressive disease after 2015 American Association for Cancer Research. initially demonstrating stable, partial, or complete response.

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Histologic confirmation of the target advanced BCC was required phy (WHO criteria; ref. 9). In cases of clinically visible BCC, where before enrollment into this study. In this study, patients required the tumor borders were complex, clean plastic grids were used to measurable, evaluable disease as defined by modified RECIST v. trace the edges of the tumors to identify the longest diameter 1.1 criteria and standard and annotated color photography (9). (Fig. 1A). Color photographs of target lesions with rulers in the Patients with basal cell nevus syndrome could enroll if they met plane of focus were taken at each clinic visit, and longest diameters inclusion criteria. identified (Fig. 1B and C). In cases where tumor borders were unclear from visual inspection of photographs, the plastic grid Exclusion criteria tracings were inspected to provide additional information on Patients were excluded from enrollment if they previously had tumor borders. Sonidegib was administered until either disease been treated with sonidegib; had completed antitumor therapy progression, as defined by RECIST v 1.1; intolerable toxicity, or less than 28 days before sonidegib initiation; or were on concur- patient or physician request to discontinue (9). Dose reduction rent antitumor therapy. was not permitted.

Study design Genetics This was an open-label, investigator-initiated study. Patients DNA from the target BCCs after vismodegib exposure, but received 800 mg oral sonidegib once daily, with treatment cycles before study entry was isolated and subjected to high-depth defined as every 28 days. This dose of sonidegib has been used in sequencing for the entire SMO coding region. Briefly, five to eight other clinical trials (7). Clinic visits and tumor assessments 10-mm sections were obtained from the formalin-fixed, paraffin- occurred every 1 to 2 treatment cycles, or sooner, if necessary, as embedded tumor block and DNA was isolated using the Qiagen deemed by the investigator. Clinic visits included medical history, DNeasy Blood and Tissue Kit according to the manufacturer's vital signs, physical examination, and adverse event recording and protocol (Qiagen). The exonic regions of SMO were amplified grading using the National Cancer Institute's Common Termi- using the Access Array platform (Fluidigm) in a multiplex format nology Criteria for Adverse Events version 4.0 (10). with genomic DNA (100 ng) according to the manufacturer's recommendation (Ambry Genetics). The multiplexed library Efficacy analysis pools were then subjected to deep sequencing using the Illumina Patients receiving greater than or equal to one dose of sonidegib MiSeq platform. FASTQ files were generated for the raw data and and having at least one follow-up tumor assessment were includ- 150 bp reads aligned to the human reference genome sequence ed in the efficacy-evaluable population. Tumor responses were (hg19) using the Burrows-Wheeler Aligner (BWA). Samtools investigator-assessed according to modified RECIST v. 1.1, which mpileup was used to call variants. Only bases meeting the min- was a composite multimodal evaluation to integrate imaging by imum base quality score of 20 from reads meeting the minimum RECIST 1.1 criteria and standard and annotated color photogra- mapping quality score of 20 were considered, and a minimum

A BC

Figure 1. Target BCC size determination of clinically visible lesions, and overall responses of enrolled subjects. Color photographs were taken of all clinically visible lesions with a ruler in the plane of the lesion. A, in cases D where the tumor borders are complex, clean plastic 60 # No SMO grids were placed over the lesions and traced to mutation with known Q477E S533N document the longest diameter. B, photograph of functional resistance 50 target lesion in a subject before study start. C, * Nonevaluable photograph of same target lesion after 6 weeks of 40 sonidegib. Arrows indicate areas of tumor growth. D, D473H D473G change in target tumor diameter from baseline (%) 30 # after sonidegib. None of the patients met the 30% partial response (top gray dotted line) per RECIST, 20 version 1.1. SMO mutations that are known to lead to functional resistance in vitro found in individual tumors 10 No are annotated for each patient. useable W535L ## 0 * tissue * –10

–20 Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Subject 7 Subject 8 Subject 9

–30 Change in lesion diameter after sonidegib (%)

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Table 1. Clinical characteristics and outcomes of 9 patients with vismodegib-resistant advanced BCCs treated with sonidegib Target tumor Target tumor SMO mutation with Sonidegib size pre- size post- known functional LA vs. Target BCC duration sonidegib sonidegib Tumor Reason for resistance to sonidegib Patient Age Sex MT BCC BCNS location (wk) (cm) (cm) response discontinuation in vitro (4, 5, 11) 1 55 M LA Paranasal 3 6.8 6.2 6.8 6.2 SD Patient election W535L for surgery 2 45 F LA X Back 3 4.0 3.0 6.0 4.0 PD PD Q477E 3 42 M MT Lymph node 58 1.0 1.0 1.0 1.3 PD PD D473H 4 51 M MT Iliac bone 5 6.4 1.2 N/A NE Grade 3 adverse event: No SMO mutations rhabdomyolysis with known functional resistance 5 91 M LA Temple 6 2.5 1.5 3.5 2.5 PD PD S533N 6 56 M LA X Tragus 4 2.5 1.8 2.0 1.7 SD Patient election No SMO mutations for surgery with known functional resistance 7 50 M MT Femoral bone 14 1.5 1.4 1.9 1.7 PD PD No SMO mutations with known functional resistance 8 59 M LA X Postauricular 12 5.0 5.0 6.4 6.0 PD PD D473G 9 68 M MT Rib 7 4.5 3.2 4.5 3.2 SD Grade 3 adverse event: Tissue unavailable nausea, altered for sequencing mental status Abbreviations: BCNS, basal cell nevus syndrome; LA, locally advanced; MT, metastatic; NE, not evaluable; PD, progressive disease; SD, stable disease. allele frequency of 5% at a position with a read depth >100 was >8,750 mg/dL, and transaminitis. All symptoms and abnormal required to make calls. SMO mutations defined as functionally blood tests resolved after intravenous fluids and discontinuation resistant mutations were those previously reported in the medical of sonidegib (10). None of the 8 evaluable patients demonstrated literature to convey resistance to SMO inhibitors in vitro (Supple- partial or complete response. The study was terminated by the mentary Table S1A; refs. 4, 5, 11). Biopsy samples of the target principal investigator due to the lack of response in any of these 9 BCCs were taken before initiation of sonidegib, but results of patients. mutational analysis were not known until after the conclusion of Adverse events while on sonidegib included grade 1–2 muscle the clinical study. cramps (N ¼ 5), grade 1 nausea (N ¼ 4), grade 1 and 3 creatine kinase elevation (N ¼ 2), grade 4 altered mental status (N ¼ 1), Statistical analysis grade 1 vomiting (N ¼ 1), grade 1 diarrhea (N ¼ 1), grade 2 weight Patient data were summarized by descriptive statistics using loss (N ¼ 1), and grade 1 loss of appetite (N ¼ 1). Microsoft Excel (version 14.5.1). The entire coding regions for SMO were sequenced, and SMO mutations with previously reported functional resistance in vitro Results to either sonidegib or vismodegib were identified in five of eight available baseline tumor samples (Table 1 and Supplementary Nine patients with an average age of 57.4 years (range, 42–91 Table S1). Four patients with an SMO mutation demonstrated years) were enrolled in the study (Table 1). Three patients had progressive disease on sonidegib, and 1 patient with an SMO basal cell nevus syndrome. Three enrolled subjects had primary mutation demonstrated stable disease for 58 weeks before even- resistance to vismodegib and 6 subjects had secondary resistance tual disease progression. The molecular structures of vismodegib to vismodegib (Supplementary Fig. S1). Of patients with second- and sonidegib bear some similarity but are chemically distinct ary resistance, 1 demonstrated prior complete response, 3 dem- (Fig. 2), but both interact with SMO at the drug-binding pocket, onstrated partial response, and 3 had stable disease. where the mutations Q477 and D473 (found in tumors from two The median duration of treatment with sonidegib was 6 weeks different subjects) are located (4, 5, 11). Two subjects' tumors had (range, 3–58 weeks). Figure 1A–C shows an example of a study mutations outside of the drug-binding pocket, S533 and W535, patient and method used to track the lesions. Overall study results and these are believed to change the conformation of SMO so that are shown in Fig. 1D. Five patients experienced progressive disease the inhibitors cannot access the drug-binding pocket (Table 1, Fig. within a median of 6 weeks (range, 3–58 weeks) of treatment with 1D, Fig. 2 and Supplementary Table S1; refs. 4, 5, 11). In addition, sonidegib, and sonidegib was discontinued, per the study proto- three subjects' BCCs possessed mutations that are not previously col (Table 1). Three patients experienced stable disease with a known to confer functional resistance in vitro: R302, H304, W549, median of 4 weeks of treatment with sonidegib (range, 3–7 Q581 R168, and N476 (Supplementary Table S1B). weeks). Two subjects elected to discontinue sonidegib for surgery. One patient elected to discontinue sonidegib due to grade 3 adverse events, including nausea and altered mental status. One Discussion subject was not evaluable due to study drug discontinuation from In this study, advanced BCCs that previously were resistant to grade 3 rhabdomyolysis secondary to sonidegib and subsequent treatment with vismodegib also were refractory to treatment with inability to travel to our clinic for a follow-up visit. Rhabdomy- sonidegib. All patients demonstrated either progressive or stable olysis was diagnosed on the basis of the overall clinical picture of disease with sonidegib. These results suggest that chemoresistance diffuse myalgias requiring narcotics, markedly dark urine, creatine can occur between different SMO inhibitors, a clinically impor- kinase elevation to 56,564 mcg/L, urine myoglobin elevation to tant finding.

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CH3

O O N CH O CH F3C 3 3 N N Q477 Drug H Figure 2. Subject 2 binding Locations of the SMO mutations known pocket to confer resistance through in vitro D473 LDE225 (sonidegib) studies were identiﬁed in the BCCs of Subjects 3, 8 the study patients. The locations of SO2Me N these mutations within the SMO protein H are shown on the left. Mutations Q476 N and D473 are within the drug-binding pocket of sonidegib. Mutations S533 Cl O and W535 likely cause conformational Cl S533 change in SMO rendering the drug- Subject 5 GDC0449 (vismodegib) binding pocket inaccessible to the sonidegib. The chemical structures of W535 sonidegib and vismodegib are shown Subject 1 on the right for comparison.

Smoothened

Clinical trials have reported an objective response in 35% of response in a phase II study has been observed in as early as 4 patients with an advanced BCC who were treated with sonidegib weeks after starting sonidegib (7). (7). This rate has similarly been reported in trials with vismodegib The population of patients who develop resistance to treatment (2). However, in these studies, patients were not previously with an SMO inhibitor is likely to increase as more patients with exposed to different SMO inhibitors, and the clinical response BCCs are treated with SMO inhibitors. Results of this study suggest rate of patients who have been previously exposed to multiple that patients with BCCs resistant to vismodegib may continue to SMO inhibitors has been unclear (2, 7). This is the first study to experience tumor progression in response to a different SMO demonstrate that patients who have been treated with a previous inhibitor. Future therapies with new targets in the hedgehog path- SMO inhibitor such as vismodegib may be resistant to treatment way downstream of SMO are needed. Combination therapy with with a different SMO inhibitor such as sonidegib. agents such as radiation, other pathway inhibitors, or immuno- SMO mutations previously identified through in vitro function- therapy may be needed to control the advanced BCC. Given the al studies as showing resistance to an SMO inhibitor were iden- advancedstateofdiseaseinmanypatients,timelyinitiationofother tified in 5 patients in this study. Given that patients with an SMO treatments besides trials of different SMO inhibitors can be critical. mutation continued to experience progressive disease on sonidegib, these mutations are likely to prevent sonidegib inhibition of Disclosure of Potential Conflicts of Interest SMO. SMO-D473 and SMO-Q477 are mutations that have been A.L.S. Chang is a consultant/advisory board member for and reports receiv- fl reported to be in the SMO binding pocket (Fig. 2; ref. 4). SMO- ing commercial research grants from Novartis. No potential con icts of interest were disclosed by the other authors. D473 (found in patient 3 and 8) was previously reported to be found in a patient with metastatic medulloblastoma and was Authors' Contributions in vitro shown to abrogate vismodegib binding (11). SMO-W535 Conception and design: C. Danial, A.E. Oro, A.L.S. Chang (patient 1) and SMO-S533 (patient 5) are mutations that have Development of methodology: C. Danial, A.E. Oro, A.L.S. Chang been reported as oncogenic drivers located outside of the binding Acquisition of data (provided animals, acquired and managed patients, pocket of SMO (Fig. 2; refs. 4, 5, 12). Interestingly, patient 3 provided facilities, etc.): C. Danial, K.Y. Sarin, A.L.S. Chang continued sonidegib for 58 weeks with stable disease before Analysis and interpretation of data (e.g., statistical analysis, biostatistics, experiencing disease progression, possibly due to tumor hetero- computational analysis): C. Danial, K.Y. Sarin, A.E. Oro, A.L.S. Chang Writing, review, and/or revision of the manuscript: C. Danial, K.Y. Sarin, A.E. geneity. Although patients with no known SMO mutation sim- Oro, A.L.S. Chang ilarly demonstrated progressive disease while on sonidegib, other Administrative, technical, or material support (i.e., reporting or organizing pathways or unknown SMO mutations may be driving tumor data, constructing databases): K.Y. Sarin, A.L.S. Chang growth. Study supervision: A.E. Oro, A.L.S. Chang A limitation of the study includes early discontinuation of sonidegib due to patient decision and adverse events. In this Grant Support study, 4 patients were treated with sonidegib for 5 weeks or less. This study was funded by Novartis. Of these 4 patients, one had progressive disease after 3 weeks of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in sonidegib, and 3 patients discontinued the study do to either side accordance with 18 U.S.C. Section 1734 solely to indicate this fact. effects or election for surgery. The short duration of sonidegib exposure in these 3 patients could have contributed to our Received July 4, 2015; revised September 23, 2015; accepted October 23, inability to detect a response, despite the fact that treatment 2015; published OnlineFirst November 6, 2015.

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References 1. Sekulic A, Migden MR, Oro AE, Dirix L, Lewis KD, Hainsworth JD, et al. 7. Migden MR, Guminski A, Gutzmer R, Dirix L, Lewis KD, Combemale P, Efﬁcacy and safety of vismodegib in advanced basal-cell carcinoma. N Engl et al. Treatment with two different doses of sonidegib in patients with J Med 2012;366:2171–9. locally advanced or metastatic basal cell carcinoma (BOLT): a multicentre, 2. Chang AL, Solomon JA, Hainsworth JD, Goldberg L, McKenna E, Day BM, randomised, double-blind phase 2 trial. Lancet Oncol 2015;16:716–28. et al. Expanded access study of patients with advanced basal cell carcinoma 8. Wang C, Wu H, Katritch V, Han GW, Huang XP, Liu W, et al. Structure of the treated with the Hedgehog pathway inhibitor, vismodegib. J Am Acad human smoothened receptor bound to an antitumour agent. Nature Dermatol 2014;70:60–9. 2013;497:338–43. 3. Chang AL, Oro AE. Initial assessment of tumor regrowth after vismodegib 9. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. in advanced Basal cell carcinoma. Arch Dermatol 2012;148:1324–5. New response evaluation criteria in solid tumours: revised RECIST guide- 4. Sharpe HJ, Pau G, Dijkgraaf GJ, Basset-Seguin N, Modrusan Z, Januario T, line (version 1.1). Eur J Cancer 2009;45:228–47. et al. Genomic analysis of smoothened inhibitor resistance in Basal cell 10. Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0. carcinoma. Cancer Cell 2015;27:327–41. 2010. Available from: http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_ 5. Atwood SX, Sarin KY, Whitson RJ, Li JR, Kim G, Rezaee M, et al. Smooth- 2010-06-14_QuickReference_8.511.pdf. ened variants explain the majority of drug resistance in Basal cell carcino- 11. Yauch RL, Dijkgraaf GJ, Alicke B, Januario T, Ahn CP, Holcomb T, et al. ma. Cancer Cell 2015;27:342–53. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor 6. Rodon J, Tawbi HA, Thomas AL, Stoller RG, Turtschi CP, Baselga J, et al. A in medulloblastoma. Science 2009;326:572–4. phase I, multicenter, open-label, ﬁrst-in-human, dose-escalation study of 12. Xie J, Murone M, Luoh SM, Ryan A, Gu Q, Zhang C, et al. Activating the oral smoothened inhibitor sonidegib (LDE225) in patients with Smoothened mutations in sporadic basal-cell carcinoma. Nature advanced solid tumors. Clin Cancer Res 2014;20:1900–9. 1998;391:90–2.

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An Investigator-Initiated Open-Label Trial of Sonidegib in Advanced Basal Cell Carcinoma Patients Resistant to Vismodegib

Christina Danial, Kavita Y. Sarin, Anthony E. Oro, et al.

Clin Cancer Res Published OnlineFirst November 6, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-15-1588

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